1. Measuring myelin protein expression with a MeCP2 knockdown in oligodendrocytes Rachel Siefring Mentor: Dr. Carmen Sato-Bigbee

2. The human population

3. People with disabilities

4. People with autism

5. ASD and Rett Syndrome Rett Syndrome Autism Spectrum Disorders

6. Cause of Rett Syndrome Caused by a mutation in methyl-CpG-binding Protein (MeCP2) Figure 2 of Cortes-Mendoza, 2013 CH 3

7. What does MeCP2 do? MeCP2 Regulates transcription of Brain-derived growth factor (BDNF) Axonal maturation Neuronal development

8. Regulates transcription of Brain-derived growth factor (BDNF) Axonal maturation Neuronal development MeCP2 What does MeCP2 do?

9. Regulates transcription of Brain-derived growth factor (BDNF) Axonal maturation Neuronal development Myelination Oligodendrocytes What does MeCP2 do? MeCP2

10. Myelination What does MeCP2 do? MeCP2

11. Myelination What does MeCP2 do? MeCP2 Figure 1B from Nave and Werner (2014) Axon

12. Myelination What does MeCP2 do? MeCP2 Oligodendrocytes Myelin Oligodendrocyte

13. Myelination Oligodendrocytes How is the myelination process different in a child with Rett syndrome? What does MeCP2 do? MeCP2

14. Progenitor cellPre-oligodendrocyte Mature oligodendrocyte Stages of oligodendrocyte development Slide provided by Dr. Sato-Bigbee

15. Myelin Proteins Myelin Basic Protein (MBP) Myelin-associated glycoprotein (MAG) Figure 1 from Jahn, et al

16. Myelin Basic Protein (MBP) MBP Modified Figure 1D from Aggarwal et al (2013)

17. Myelin-associated glycoprotein (MAG) Acts as a linker between the axon and the myelin sheath

18. Is there a difference in MBP and MAG expression levels when there is a MeCP2 knockdown??

19. Isolating oligodendroctyes Sprague-Dawley rats 2 experimental groups Regular MeCP2MeCP2 knockdown From Figure 1A from Nguyen et al (2013) Cerebral hemispheres and cerebellum

20. Knockdown method: siRNA

21. siRNA synthesis Send MeCP2 sequence to Sigma Aldrich 300-600 nt mRNA transcript Use DEQOR to generate 21 nucleotide transcripts Synthesized siRNA

22. Applying siRNA to oligodendrocytes siRNA GeneJammer reagent See if knockdown worked via Western Blot

23. Western Blot: SDS-PAGE Make cell lysates for each culture Load samples into the SDS-PAGE Separated lysate proteins

24. Western Blot http://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Western_Blotting Polymer sheet is exposed to antibodies

25. Western Blot Figures 7A and 7B from Nguyen (2013)

26. Possible resulting scenarios Methods don’t work siRNA Oligodendrocyte isolation fails

27. Possible resulting scenarios Methods don’t work siRNA Oligodendrocyte isolation fails No difference in myelin protein expression MeCP2 doesn’t effect oligodendrocytes in culture

28. Possible resulting scenarios Methods don’t work siRNA Oligodendrocyte isolation fails No difference in myelin protein expression MeCP2 doesn’t effect oligodendrocytes in culture Large differences in myelin protein expression MeCP2 does effect oligodendrocytes in culture Underexpression Overexpression

29. Concluding remarks Do myelination and oligodendrocytes play a role in the Rett syndrome phenotype? Does MeCP2 specifically affect MBP and MAG? Based on this experiment, should this research even be pursued? Does MeCP2 directly affect the myelination process?

30. References: 1.Bhat, S., et al. (2014). Autism: cause factors, early diagnosis, and therapies. Reviews in the Neurosciences. De Gruyter. 25(6): 841- 850. 2.Amir, R. et al. (1999). Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature Genetics, 23: 185-188. 3.Kleijer, K., Schmeisser, M., Krueger, D., Boeckers, T., Scheiffele, P., Bourgeron, T., Brose, N., and J. Burbach. 2014. Neurobiology of autism gene products: towards pathogenesis and drug targets. Psychopharmacology. 231: 1037-1062. 4.Chahrour, M., et al. (2008). MeCP2, a key contributor to neurological disease, activates and represses transcription. Science, 320: 1224-1229. 5.Ebert, D. & M. Greenberg. (2013). Activity-dependent neuronal signalling and autism spectrum disorder. Nature, 493: 327-337. 6.Vora, P., et al. (2010). A novel transcriptional regulator of myelin gene expression: implications for neurodevelopmental disorders. Lippincott Williams & Wilkins, 0959-4965: 917-921. 7.Zeidán-Chuliá, F., et al. (2014). The glial perspective of autism spectrum disorders. Neuroscience and Biobehavioral Reviews, 38: 160-172.

31. References, cont. 8. Karpova, N. (2014). Role of BDNF epigenetics in activity-dependent neuronal plasticity. Neuropharmacology, 76: 709-718. 9. Cortes-Mendoza, J., et al. (2013). Shaping synaptic plasticity: the role of activity-mediated epigenetic regulation on gene transcription. International Journal of Developmental Neuroscience. 31: 359-369. 10. Sato-Bigbee, C., et al. (1999). Different neuroligands and signal transduction pathways stimulate CREB phosphorylation at specific developmental stages along oligodendrocyte differentiation. Journal of Neurochemistry, 72: 139-147. 11. Saini, H., et al. (2005). Novel role of sphinogosine kinase 1 as a mediator of neurotrophin-3 action in oligodendrocyte progenitors. Journal of Neurochemistry, 95: 1298- 1310. 12.Shan, G. (2010). RNA interference as a gene knockdown technique. The International Journal of Biochemistry and Cell Biology, 42: 1243-1251. 13.Nguyen, M., et al. (2013). Oligodendrocyte lineage cells contribute unique features to Rett Syndrome neuropathology. Neurobiology of Disease, 33(48): 18764- 18774. 14.Bradl, M. and H. Lassmann. (2010). Oligodendrocytes: biology and pathology. Acta Neuropathol, 119: 37-53. 15.Jahn, O., Tenzer, S., and H. Werner. (2009). Myelin proteomics: molecular anatomy of the insulating sheath. Mol Neurobiol, 40: 55-72.